Project Summary Generation and Biophysical Evaluation of Generic Fab Antibody pH Switches Antibody-antigen interactions have served as a prototypical model for better understanding protein-protein interactions and protein design, as well as serving a vital role in life science applications. Such antibodies are indispensable to a wide range of fields including cell and molecular biology, molecular imaging, diagnostics, nanotechnology, and next generation antibody therapeutics. While the goal of antibody engineering is most often focused on achieving high affinity or stability, over the last several years, the Horn lab has explored new directions in the design of protein interactions, namely the introduction of linked equilibria that may be used to control (or regulate) antibody/antigen interactions. These efforts successfully produced antibody variants with gain-of-function pH switches, allowing the protein interaction to be modulated over a range in pH. This proposal aims to explore a new twist on these efforts by investigating methods to indirectly control conventional antibody interactions through pH-based control of the antibody?s VH/VL interface. This research has the potential to identify antibody framework residues that may be used as generic pH-based triggers to control the binding event of almost any antibody/antigen system. Given the wide-spread use of antibodies in applications where pH-based control provides functional advantages, the outcomes of this work are likely to be of wide interest.